BLT2 up-regulates interleukin-8 production and promotes the invasiveness of breast cancer cells.

1School of Life Sciences and Biotechnology, Korea University, Seoul, Korea.

Abstract

BACKGROUND:

The elevated production of interleukin (IL)-8 is critically associated with invasiveness and metastatic potential in breast cancer cells. However, the intracellular signaling pathway responsible for up-regulation of IL-8 production in breast cancer cells has remained unclear.

METHODOLOGY/PRINCIPAL FINDINGS:

In this study, we report that the expression of BLT2 is markedly up-regulated in the highly aggressive human breast cancer cell lines MDA-MB-231 and MDA-MB-435 compared with MCF-10A immortalized human mammary epithelial cells, as determined by RT-PCR, real-time PCR and FACS analysis. Blockade of BLT2 with BLT2 siRNA knockdown or BLT2 inhibitor treatment downregulated IL-8 production and thereby diminished the invasiveness of aggressive breast cancer cells, analyzed by Matrigel invasion chamber assays. We further characterized the downstream signaling mechanism by which BLT2 stimulates IL-8 production and identified critical mediatory roles for the generation of reactive oxygen species (ROS) and the consequent activation of the transcription factor NF-κB. Moreover, blockade of BLT2 suppressed the formation of metastatic lung nodules by MDA-MB-231 cells in both experimental and orthotopic metastasis models.

CONCLUSIONS/SIGNIFICANCE:

Taken together, our study demonstrates that a BLT2-ROS-NF-κB pathway up-regulates IL-8 production in MDA-MB-231 and MDA-MB-435 cells, thereby contributing to the invasiveness of these aggressive breast cancer cells. Our findings provide insight into the molecular mechanism of invasiveness in breast cancer.

(A and C) Semiquantitative RT-PCR (left panel) and quantitative real-time PCR (center and right panels) analysis of BLT1 and BLT2 mRNAs in MCF-10A (control) as well as MDA-MB-231 (A) and MDA-MB-435 (C) cells. Data are representative of three independent experiments. (B and D) Flow cytometric analysis of surface BLT2 expression. All quantitative data are expressed relative to the value for MCF-10A cells and are means±SD from three independent experiments. *P<0.05.

BLT2 is required for the invasiveness of MDA-MB-231 and MDA-MB-435 breast cancer cells.

(A and B) MDA-MB-231 (A) and MDA-MB-435 (B) cells were transfected with BLT2 (siBLT2) or control (siCont) siRNAs for 48 h in medium supplemented with 0.5% FBS, after which the amounts of BLT1 and BLT2 mRNAs were determined by semiquantitative RT-PCR (left panel) and quantitative real-time PCR (center and right panels) analysis. Data are representative of five independent experiments. (C) Cells treated as in (A) and (B) were assayed for invasiveness. Representative invading cells stained with H&E are shown, and quantitative data are expressed relative to the value for cells transfected with the control siRNA. Scale bars, 50 µm. (D) MDA-MB-231 and MDA-MB-435 cells were incubated with U75302 (0.5 or 1 µM), LY255283 (5 or 10 µM), or DMSO vehicle for 30 min and were then assayed for invasiveness in the continued presence of the corresponding test agent. Scale bars, 50 µm. All quantitative data are means±SD from five independent experiments. *P<0.05, †P<0.01, ‡P<0.005.

ROS production occurs downstream of BLT2 and contributes to the invasiveness of breast cancer cells.

(A) MCF-10A, MDA-MB-231, and MDA-MB-435 cells were incubated in the presence of 0.5% serum for 3 h and then loaded with the diacetate form of DCF (20 µM) for 20 min. Intracellular ROS levels were then determined by flow cytometric analysis of DCF fluorescence. (B) MDA-MB-231 and MDA-MB-435 cells were incubated with U75302 (1 µM), LY255283 (10 µM), or DMSO vehicle for 1 h before determination of the intracellular level of ROS as in (A). (C and D) MDA-MB-231 and MDA-MB-435 cells were incubated with NAC (5 mM), DPI (0.5 µM), or DMSO vehicle for 1 h and then monitored for ROS levels (C) or assayed for invasiveness in the continued presence of the test agents (D). All data are expressed relative to the corresponding control value and are means±SD from five independent experiments. *P<0.05, †P<0.01, ‡P<0.005.

(A) Semiquantitative RT-PCR analysis of Nox1 and Nox4 mRNAs in MCF-10A, MDA-MB-231, and MDA-MB-435 cells. Data are representative of three independent experiments. (B) MDA-MB-231 and MDA-MB-435 cells were incubated with U75302 (1 µM), LY255283 (10 µM), or DMSO vehicle for 1 h or were transfected with BLT2 or control siRNAs for 48 h before determination of the abundance of Nox1 and Nox4 mRNAs by semiquantitative RT-PCR analysis. Data are representative of three independent experiments. (C and D) MDA-MB-231 and MDA-MB-435 cells were transfected with a vector for Nox1 siRNA (pSUPER-siNox1) or the corresponding empty vector for 48 h and were then analyzed for the intracellular level of ROS (C) or invasiveness (D). Scale bars, 50 µm. Data are expressed relative to the corresponding value for cells transfected with the empty vector and are means±SD from five independent experiments. *P<0.05, †P<0.01, ‡P<0.005.

(A–C) MDA-MB-231 and MDA-MB-435 cells were incubated with U75302 (1 µM), LY255283 (10 µM), or DMSO vehicle for 6 h (A), were transfected with BLT2 or control siRNAs for 48 h (B), or were incubated with NAC (5 mM), DPI (0.5 µM), or DMSO for 6 h (C). Cell lysates were then subjected to immunoblot analysis with antibodies to phosphorylated IκB-α (p-IκB-α) and to α-tubulin (loading control). Numbers under lanes indicate relative density of p-IκB-α from three independent experiments. (D and E) MDA-MB-231 and MDA-MB-435 cells were transiently transfected with control or BLT2 siRNAs and the pNF-κB-Luc reporter plasmid for 24 h, deprived of serum for 12 h, and then harvested (D). Alternatively, the cells were transiently transfected with pNF-κB-Luc, incubated with NAC (5 mM), DPI (0.5 µM), or DMSO for 6 h, and harvested (E). All cells were then assayed for relative luciferase activity. Data are expressed relative to the corresponding control value and are means±SD from three independent experiments. *P<0.05, †P<0.01, ‡P<0.005.

BLT2-dependent production of IL-8 contributes to the invasiveness of breast cancer cells.

(A) MCF-10A, MDA-MB-231, and MDA-MB-435 cells were subjected to semiquantitative RT-PCR analysis of IL-8 mRNA (left panels). The cells (2×105) per well in six-well plates were also incubated for 48 h, after which the amount of IL-8 released into the medium was measured with the use of a specific ELISA (right panel). (B) MDA-MB-231 and MDA-MB-435 cells were transfected with BLT2 or control siRNAs for 48 h, after which the secretion of IL-8 was determined as in (A). (C) MDA-MB-231 and MDA-MB-435 cells were incubated with Bay11-7082 (10 µM) or DMSO vehicle for 48 h, after which the amount of IL-8 released into the medium was determined. (D) MDA-MB-231 and MDA-MB-435 cells were transfected with a vector for Nox1 siRNA (pSUPER-siNox1) or the corresponding empty vector for 48 h, after which the secretion of IL-8 was determined as in (A). (E and F) MDA-MB-231 and MDA-MB-435 cells were transfected with sense (S) or antisense (AS) IL-8 oligonucleotides for 48 h, after which the amounts of IL-8 were determined by semiquantitative RT-PCR and ELISA assays (E) and the cells were assayed for invasiveness (F). Scale bars, 50 µm. Semiquantitative RT-PCR data are representative of three independent experiments and all quantitative data are means±SD from three independent experiments. *P<0.05, †P<0.01, ‡P<0.005.

(A and B) MDA-MB-231 cells treated for 24 h with LY255283 (10 µM) or DMSO were injected into the tail vein of nude mice, which were subsequently injected intraperitoneally with DMSO (control) or LY255283 (2.5 mg/kg) at 3 and 5 days after cell injection. Mice were killed 10 weeks after cell injection, and the lungs were removed and subjected to histological analysis (A). Arrowheads indicate metastatic nodules. Scale bars, 5 mm (upper panels) and 200 µm (lower panels). The number of metastatic nodules in the lungs was also counted (B); data are means±SD of values from four mice of each group. *P<0.05. (C and D) MDA-MB-231 cells pretreated for 24 h with LY255283 (10 µM) or DMSO were implanted into the mammary fat pad of nude mice, and the animals were then injected intraperitoneally with LY255283 (2.5 mg/kg) or DMSO (control) three times at 5-day intervals. The mice were killed 14 weeks after cell implantation, and lung sections were stained with H&E for analysis of metastatic nodules (C). Scale bars, 200 µm. The number of metastatic nodules in the lung was also counted (D). Data are representative of results obtained with three mice per group. *P<0.05.

Scheme for the involvement of BLT2 cascade in IL-8 production and invasiveness of breast cancer cell.

BLT2-induced IL-8 up-regulation leads to enhanced invasiveness of breast cancer cells. The activation of the BLT2 pathway induced Nox1-dependent ROS generation. Then, these ROS may contribute to NF-κB activation, which lead to IL-8-dependent invasiveness.